Connective tissue and Muscle staining

Define connective tissue and its function

It is a fibrous material

Functions: support and protection, a media for exchange of nutrients, energy storage, insulation, and tissue repair

Three main components of CT

cells, fibers and extracellular matrix or ground substance

Most abundant fiber found in CT

Collagen

Define collagens make up and function

-Tough, thick, fibrous proteins that do not branch

-Provides great tensile strength giving the body form, firmness and resistance to outside forces

Staining characteristics

• They are cationic (basic or positively charged)

• Stain strongly with acid dyes (anionic or negatively charged)

• Eosinophilic

• Strongly birefringent under polarized light

• Demonstrated most frequently with the Masson or Gomori Trichrome staining techniques

Diagnostic application of staining for CT

Plays a role in diagnosing collagenous CT diseases (cirrhosis and kidney-glom)

Plays a role in diagnosing soft tissue - tumors, cardiovascular

Most importantly- distinguishes muscle from CT

Mass Trichrome purpose

 To identify an increase in collagenous connective tissue fibers such as in cirrhosis of the liver or to differentiate between collagen and muscle fibers

Mass Trichrome fixative, control tissue and microns

Fixative: Bouin, 10% NBF

Control tissue: Uterus, small intestine, appendix, or fallopian tube

Microns: 4-5

Masson Trichrome reagents and their purpose

Bouin	Mordant
Weigert’s Hematoxylin	Nuclear Counterstain
Biebrich Scarlet	Stains cytoplasm, collagen and muscle
Phosphotungstic and/or  phosphomolybdic acid	Differentiation
Aniline Blue	Stains collagen
1% Acetic Acid	Differentiation

Masson Trichrome key quality Indicators

Key Quality Indicators:

• Distinct blue/black nuclei

• Strongly stained, brilliant blue or green collagen and red muscle

• Excellent contrast between the collagen and muscle

• Even staining throughout tissue section

• Clean background, free of staining artifacts (e.g. precipitate, background staining)

Masson trichrome color results

Brilliant red… Cytoplasm, Keratin, Muscle fibers, Intercellular fibers, RBC’s

Blue…Collagen and mucin

Nuclei…… Black

Movat Pentchrome purpose

To demonstrate mucin, fibrin, elastic fibers, muscle, and collagen

Movat Pentachrome Fixative, thickness and control tissue

Fixative: 10% NBF

Thickness: 4-5 microns

Control Tissue: Lung, skin, aorta

Movat Pentachrome Reagents and their purpose

Alcian Blue 2.5	Stains acid mucosubstances
Alkaline Alcohol	Converts Alcian blue into insoluble monastral fast blue
Verhoeff Working Solution	Stains elastic fibers
2% Ferric Chloride	Differentiation
5% Sodium Thiosulfate	Removes residual iodine
Crocein scarlet-acid fuchsin working solution	Stains muscle and fibrin
0.5% Acetic Acid	Lowers pH and safeguards phosphotungstic acid solution
5% Phosphotungstic Acid	Differentiation
0.5% Acetic Acid	Removes excess phosphotungstic acid
Absolute Alcohol	Removes excess acid and dehydrates
Alcoholic Spanish Saffron	Stains collagen and reticular fibers (counterstain)

Movat Pentachrome Color results

Nuclei and Elastic fibers	Black
Collagen and Reticular fibers	Yellow
Ground substance, Mucin	Blue
Fibrinoid, Fibrin	Intense red
Muscle	Red
C. Neoformans	Bright Blue

Movat Pentachrome Key quality Indicators

• Strongly stained, well demonstrated carbohydrates (mucin) and elastic fibers

• Counterstain that enhances the desired structures

• Excellent contrast between all structures to be demonstrated

• Clean background, free of staining artifacts (e.g. precipitate, background staining)

Reticulin Purpose

Reticulin Fixative, Thickness, and control tissue

Fixative: 10% NBF

Thickness: 4-5 microns
Control: Liver

Reticulin Reagents and their purpose

Acetified Potassium Permanganate	Oxidation to aldehydes
1% Oxalic Acid	Bleaches potassium permanganate
2% Ferric Ammonium Sulfate	Sensitizer
Diamine Silver	Impregnation
10% Formalin	Reduces silver to a visible metallic form (develop)
0.2% Gold Chloride	Tones silver
5% Sodium Thiosulfate	Removes unreduced silver
Nuclear Red Fast	Nuclear counterstain

Reticulin color Results

Reticular fibers	Black
Background	Pink

Reticulin Key Quality Indicators

• Well defined, linear (rather than a granular) pattern of reticular fibers

• Strongly stained black reticular fibers

• Counterstain enhances reticular fibers

• Nuclear staining the color of the counterstain (not the silver)

• Clean background, free of staining artifacts (e.g. precipitate, nonspecific or background staining)

8 steps of silver

1. Oxidation

2. Removal of excess potassium permanganate

3. Sensitization

4. Impregnation

5. Reduction

6. Tone

7. Remove unreacted silver

8. Counterstain (optional)

Three theories of staining method for Trichrome

1. Size of dye molecule (diffusion)

2. Varying affinity of anionic dyes to cationic tissue components

3. Mordants

Size of dye molecule theory- trichrome

Biebrich scarlet is relatively small and can therefore penetrate into the compact spaces within muscle fibers

Aniline blue is relatively large and can only penetrate into the more open spaces of connective tissue

Mordant theory- trichrome

Phosphotungstic and Phosphomolybdic act as mordants in binding aniline blue to the collage

Affinity theory- Trichrome

Biebrich scarlet and aniline blue are believed to have varying affinity for the various tissue components

Why is Bouin used in Masson Trichrome

Mordant - enhance the staining intensity and brilliancy of the subsequent colors

What type of dye is Biebrich and what tissue is it affinity for

Acid dye- will dye acidophilic tissues (cytoplasm, muscle, and collagen)

Which tissue region does phosphotungstic cause Biebrich to diffuse out of and why?

Biebrich will diffuse out of the collagen but no the cytoplasm or muscle because collagen has higher permeability (looser configuration)

Phosphotungstic and phosphomolybdic acids are mordants

True - acidic properties that act as a link (mordant) between the decolorized collagen and aniline blue

How does the pH of phosphotungstic and phosphomolybdic acids affect staining

Increases selective collagen staining and aids in the diffusion or removal of Biebrich scarlet allowing for more binding sites for the aniline blue

Analine blue is _______ charged and is ______ in size, allowing for it to permeate collagen

Negatively, larger

Gomori One-Step Trichrome purpose

Identify an increase in collagenous connective tissue fibers in diseases such as cirrhosis of the liver or to differentiate between collagen and muscle fibers.

Same as Masson

Gomori One-Step Trichrome reagents and their purpose

Define elastic fibers

Thin, small, branching fibers that allow for stretching and flexibility of tissue

Found in CT - lungs, skin and the cardiovascular system

Which tissue component has less tensile strength than collagen and are composed of microfibrils and the protein elastin

Elastic fibers

Diagnostic purposes of staining for elastic fibers (4)

Atrophy of elastic fibers can be an indicator of emphysema

Thinning or loss of fibers can be an indicator of atherosclerosis

Reduplication, breaks, or splitting can be an indicator of vascular disease

Tumors can also be detected in the blood vessels by staining for fibers

VVG demonstrates

Detect changes in elastic fibers

VVG fixative, thickness and control tissue

Fixative: 10% NBF, zenker

Thickness: 4-5 microns

Control: Aorta on edge, cross section of artery

VVG reagents and their purpose

Verhoeff Working Solution	Stains elastic fibers and nuclei
2% Ferric Chloride	Differentiation
95% Alcohol	Prepares slide for Van Gieson
Van Gieson	Stains collagen, muscle and cytoplasm
Methanol	Dehydration
Acetone	Dehydration
Acetone	Dehydration

VVG color results

Elastic fibers.....blue-black to black
Nuclei.....blue to black
Collagen.....red
Other tissue elements.....yellow

Verhoeff Elastic solution is a _____ dye lake that consists of what reagents

soluble

Hematoxylin, ferric chloride and iodine

What are the two mordants in Verhoeff Elastic solution?

What other purpose do they serve?

Iodine and ferric chloride

They are also oxidizers that convert hematoxylin to hematein

How does Verhoeff Elastic solution dye bind to the tissue

formation of hydrogen bonds between the tissue and dye

Is VVG regressive or progressive? why?

Regressive- overstained with Verhoeff Elastic solution then differentiated with an excess mordnat, ferric chloride

Van Gieson in VVG is made up of what reagents

picric acid and acid fuchsin

How does binding work with picric acid and acid fuchsin in a VVG

small molecules of picric acid penetrate all of the tissues rapidly, but are only firmly retained in the close textured red blood cells and muscle

The larger molecules of acid fuchsin (an acidic aniline dye) displace picric acid molecules from collagen fibers, which have larger pores, and allow the larger molecules to enter.

What’s important that is provided by picric acid in Van Gieson

Low pH- allows for selective staining of collagen

What happens if you prolong staining in Van Gieson

picric acid can differentiate the elastic fibers stain

Why is the dehydration run down different in VVG than standard staining methods

Dehydrated- methanol

Cleared- acetone

Regular run down can wash the dye out (decolorize) of the tissue

Define reticular fibers

a type of thin, branching connective tissue fiber composed of type III collagen- creates a mesh network- found in the liver, lymph nodes, spleen, and bone marrow

Diagnostic applications of reticular fibers - how are they demonstrated in carcinomas, sarcomas and lymphosarcomas

Tumor identification, differential diagnosis of certain tumor types and any changes in reticular fiber patterns

Carcinomas- reticulum surrounds nests of tumor cells and supports the outer surface

Sarcomas- mesh-like pattern is demonstrated with each cell surrounded by some reticulum

Lymphosacrcomas- fibers can be found between individual cells within lymph node

Reticulin demonstrates

Demonstration of reticular fibers

Reticulin reagents and their purpose

Acetified Potassium Permanganate	Oxidation to aldehydes
1% Oxalic Acid	Bleaches potassium permanganate
2% Ferric Ammonium Sulfate	Sensitizer
Diamine Silver	Impregnation
10% Formalin	Reduces silver to a visible metallic form (develop)
0.2% Gold Chloride	Tones silver
5% Sodium Thiosulfate	Removes unreduced silver
Nuclear Red Fast	Nuclear counterstain

Reticulin color results

Reticular fibers	Black
Background	Pink

Reticulin Fixative, thickness and control tissues

Fixative: 10% NBF

Thickness: 4-5

Controls: Liver

8 steps of staining with sliver stains

1. Oxidation

2. Removal of excess potassium permanganate

3. Sensitization

4. Impregnation

5. Reduction

6. Tone

7. Remove unreacted silver

8. Counterstain (optional)

Walk through the oxidation step of reticulin

Glycol groups of the hexose sugars in the reticular fibers are first oxidized to aldehydes by acetified potassium permanganate

Why is acetified potassium permanganate used as the oxidizer for reticulin

It prevents silver staining of the nuclei

What is used to remove or “bleach” excess potassium permanganate in Retic

Oxalic acid

Walk through the sensitizer step of Retic

Ferric ammonium sulfate, due its low affinity to silver, it enhances reticular staining- forms a metal organic compound with the tissue

Walk through the impregnation step in Retic

Diamine silver replaces the metal organic compound made by ferric ammonium sulfate - the deposition of silver or gold ON or around the tissue, not in the tissue

What can cause the tissue to detach from the slide in a retic stain, how can it be avoided

Diamine silver is alkaline and has a pH of 11-12- the basicity of the dye can cause the tissue to detach

Silanized, charged, or coated slides may need to be used to avoid it

Walk through the reduction step of retic - differentiate argyrophill and argentaffin

Formalin is used as a reducing agent to reduce or developed the silver that has been deposited

Argyrophill - an external reagent is used as a reducer (formalin)

Argentaffin - Stains that does not require an external reducer

Walk through the toning step of Retic- why is this step important

Gold chloride is used - toning is the process when the metallic silver is replaced with metallic gold

allows for clarity and better contrast of reticular fibers - gold is also more stable

How do we remove unreacted silver in Retic

Sodium thiosulfate removes any unreacted silver that remains in the tissue to prevent any nonspecific reduction of silver by natural light

Define fibrin

a fibrous protein involved in blood clotting that is polymerized to create a “mesh” that forms a plug or clot over a wound site- seen in tissue with tissue damage or acute inflammation

Diagnostic applications of staining for fibrin

Excess fibrin can lead to thrombosis= blood clot

Disease of the liver can decrease fibrin production

The consequences of reduced, absent, or dysfunctional fibrin is likely to render patients as hemophiliacs

Movat Pentachrome demonstrates

To demonstrate mucin, fibrin, elastic fibers, muscle, and collagen

Movat Pentachrome thickness, fixative and control tissue

Fixative: 10% NBF
Thickness: 4-5 microns
Control tissue: Lung, skin, aorta

Movat Pentachrome reagents and their importance

Alcian Blue 2.5	Stains acid mucosubstances
Alkaline Alcohol	Converts Alcian blue into insoluble monastral fast blue
Verhoeff Working Solution	Stains elastic fibers
2% Ferric Chloride	Differentiation
5% Sodium Thiosulfate	Removes residual iodine
Crocein scarlet-acid fuchsin working solution	Stains muscle and fibrin
0.5% Acetic Acid	Lowers pH and safeguards phosphotungstic acid solution
5% Phosphotungstic Acid	Differentiation of crocein scarlet acid fuchsin WS
0.5% Acetic Acid	Removes excess phosphotungstic acid
Absolute Alcohol	Removes excess acid and dehydrates
Alcoholic Spanish Saffron	Stains collagen and reticular fibers (counterstain)

Movat Pentachrome color results

Nuclei and Elastic fibers	Black
Collagen and Reticular fibers	Yellow
Ground substance, Mucin	Blue
Fibrinoid, Fibrin	Intense red
Muscle	Red
C. Neoformans	Bright Blue

Define lipid

Insoluble in water

Function as structural components of cell membranes, serve as a metabolic energy reserve and protect numerous organs

Simple lipids which include fatty acids, waxes, triglycerides and sterols

Diagnostic application of staining for lipids

Fixative used for lipids

Osmium tetroxide, uncommon so we opt for frozen sections

Oil red O demonstrates

To demonstrate neutral (non-charged) lipids

Oil red O reagents and their purpose

10% NBF	Fixation
50% Isopropyl alcohol	Dehydration
Oil Red O	Stains fat
50% Isopropyl alcohol	Prevents nonspecific staining
Hematoxylin	Nuclear stain
Saturated Lithium Carbonate	Bluing

Oil Red O color results

Fat	Red
Nuclei	Blue

Oil Red O Fixative, Thickness and control

Fixative: None (frozen)

Thickness: 10 microns
Control: skin

Is Oil Red O a physical method of staining or is it a chemical method- what does this mean?

PHYSICAL - physically moves out of the staining solution and into the fat

What four features should a stain have to stain with fat

1. Be more soluble in the tissue lipid than in the solvent in which it is dissolved

2. Must not be water soluble

3. Must be strongly colored

4. Must act with tissue constituents only by solution. Note: the solvent used is critical, with isopropanol removing a minimal amount of lipid and propylene glycol not extracting any lipid.

What are two dyes that work similarly to Oil Red O

Sudan IV (solvent 70%) and Sudan Black B (solvent propylene glycol)

What mounting media is used with Oil Red O

Aqueous mounting media

What cells are found in connective tissue (8)

Diagnostic application of staining for connective tissue

Mast and plasma cells, components of the immune system, are indicative of disease

Mast cells are metachromatic

true - they will stain a different color from the dye solution and the rest of the tissue

TBO demonstrates

Mast cells

TBO reagents and their purpose

Toluidine Blue Solution	Stains mast cells
0.2% acetic acid	Differentiation

TBO color results

Mast cells	Deep Violet
Background	Blue
Nuclei, Nissl granules	Dark blue
Cytoplasm, muscle	Light blue
Mucin	Red/purple
Red blood cells	Green

TBO fixative, thickness and control tissue

Fixative: 10% NBF
Thickness: 4-5 microns
Controls: Mast cell sections (Spleen, liver)

Define Basement membranes

provide structural support to the epithelium and act as a barrier from the underlying connective tissue

Diagnostic applications of staining basement membranes

Important for kidney disease- basement membrane of gloms

Periodic Acid Methenamine demonstrates

To demonstrate glomerular basement membranes in kidney

Periodic Acid Methenamine reagents and their purpose

1% Periodic acid	Oxidation to aldehydes
Methenamine Silver	Impregnation
0.2% Gold chloride	Tones silver
2% Sodium Thiosulfate (Hypo)	Removes unreduced silver
Light Green	Counterstain

Periodic Acid Methenamine color results

Basement membrane	Black
Background	Green

Periodic Acid Methenamine Fixative, Thickness and controls

Fixative: 10% NBF (avoid mercury- Zenker and B-5)

Thickness: 2 microns

Controls: Kidney